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Guanylate-Binding Protein 1 (GBP1) Enhances IFN-α Mediated Antiviral Activity against Hepatitis B Virus Infection

Yadi Li
Yadi Li
, 
Haiying Luo
Haiying Luo
, 
Xiaoxia Hu
Xiaoxia Hu
, 
Jiaojiao Gong
Jiaojiao Gong
, 
Guili Tan
Guili Tan
, 
Huating Luo
Huating Luo
, 
Rui Wang
Rui Wang
, 
Hao Pang
Hao Pang
, 
Renjie Yu
Renjie Yu
 und 
Bo Qin
Bo Qin
   | 20. Juni 2024
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Article Category: Original Paper
Online veröffentlicht: 20. Juni 2024
Seitenbereich: 217 - 235
Eingereicht: 07. Feb. 2024
Akzeptiert: 08. Mai 2024
DOI: https://doi.org/10.33073/pjm-2024-021
Schlüsselwörter
© 2024 Yadi Li et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

HBV infection upregulated GBP1 expression. A) The levels of HBV total RNA and GBP1 mRNA were determined by qRT-PCR in uninfected and infected HepG2-NTCP cells; B) HepG2-NTCP cells uninfected and infected were analyzed by Western blot for GBP1 and HBs expression; C–D) changes in the expression of HBV total RNA (C) and GBP1 mRNA (D) at different times of HBV infection. E-F) changes in the expression of GBP1 and HBs proteins at different times of HBV infection. Data are presented as means ± SD; ***p < 0.001. Data are presented as means ± SD; ***p < 0.001.
HBV infection upregulated GBP1 expression. A) The levels of HBV total RNA and GBP1 mRNA were determined by qRT-PCR in uninfected and infected HepG2-NTCP cells; B) HepG2-NTCP cells uninfected and infected were analyzed by Western blot for GBP1 and HBs expression; C–D) changes in the expression of HBV total RNA (C) and GBP1 mRNA (D) at different times of HBV infection. E-F) changes in the expression of GBP1 and HBs proteins at different times of HBV infection. Data are presented as means ± SD; ***p < 0.001. Data are presented as means ± SD; ***p < 0.001.

Fig. 2.

GBP1 potentiated the anti-viral function of IFN-α. A) Contents of HBsAg and HBeAg were determined by ELISA in HBV-infected HepG2 2.15 and HepG2-NTCP cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. B–C) relative changes in the levels of HBV total RNA (B) and GBP1 mRNA (C) in HBV-infected cells; D–E) levels of GBP1 (D) and HBs (E) proteins were examined in HBV-infected cells. Data are presented as means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
GBP1 potentiated the anti-viral function of IFN-α. A) Contents of HBsAg and HBeAg were determined by ELISA in HBV-infected HepG2 2.15 and HepG2-NTCP cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. B–C) relative changes in the levels of HBV total RNA (B) and GBP1 mRNA (C) in HBV-infected cells; D–E) levels of GBP1 (D) and HBs (E) proteins were examined in HBV-infected cells. Data are presented as means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3.

HBV infection prolonged the half-life of GBP1 protein. A-B) Relative changes in the levels of HBV total RNA (A) and GBP1 mRNA (B) in HBV-infected and stimulated HepG2 2.15 cells with Peg-IFNα-2b for 96 hours; C) levels of GBP1 protein were examined in HBV-infected HepG2 2.15 cells and stimulated with Peg-IFNα-2b for 96 hours; D–E) levels of GBP1 protein were determined in uninfected and infected HepG2-NTCP cells treated with cycloheximide (CHX). Data are presented as means ± SD.
HBV infection prolonged the half-life of GBP1 protein. A-B) Relative changes in the levels of HBV total RNA (A) and GBP1 mRNA (B) in HBV-infected and stimulated HepG2 2.15 cells with Peg-IFNα-2b for 96 hours; C) levels of GBP1 protein were examined in HBV-infected HepG2 2.15 cells and stimulated with Peg-IFNα-2b for 96 hours; D–E) levels of GBP1 protein were determined in uninfected and infected HepG2-NTCP cells treated with cycloheximide (CHX). Data are presented as means ± SD.

Fig. 4.

GBP1 silencing promoted HBV replication. A–C) HepG2 2.15 cells transduced with specific siRNAs (si-control, si-GBP1-1, and si-GBP1-2) silence endogenous GBP1: ELISA kits (A), qRT-PCR (B), and Western blot (C) were employed to examine the indicated genes expression; D–F) before being gathered, HepG2 2.15 cells were transfected with the wild-type hGBP1 plasmid or si-GBP1, and followed by stimulation with IFN-α (1,000 U/ml): ELISA kits (D), qRT-PCR (E), and Western blot (F) were employed to examine the indicated genes expression. Data are presented as means ± SD; *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001. D-F) before being gathered, HepG2 2.15 cells were transfected with the wild-type hGBP1 plasmid or si-GBP1, and followed by stimulation with IFN-α (1,000 U/ml): ELISA kits (D), qRT-PCR (E), and Western blot (F) were employed to examine the indicated genes expression; G–I) transfected HepG2-NTCP cells were treated with 1,000 U/ml IFN-α for 48 h after transfection with wild-type or si-GBP1 plasmids: ELISA kits (G), qRT-PCR (H), and Western blot (I) were employed to examine the indicated genes expression. Data are presented as means ± SD; *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001.
GBP1 silencing promoted HBV replication. A–C) HepG2 2.15 cells transduced with specific siRNAs (si-control, si-GBP1-1, and si-GBP1-2) silence endogenous GBP1: ELISA kits (A), qRT-PCR (B), and Western blot (C) were employed to examine the indicated genes expression; D–F) before being gathered, HepG2 2.15 cells were transfected with the wild-type hGBP1 plasmid or si-GBP1, and followed by stimulation with IFN-α (1,000 U/ml): ELISA kits (D), qRT-PCR (E), and Western blot (F) were employed to examine the indicated genes expression. Data are presented as means ± SD; *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001. D-F) before being gathered, HepG2 2.15 cells were transfected with the wild-type hGBP1 plasmid or si-GBP1, and followed by stimulation with IFN-α (1,000 U/ml): ELISA kits (D), qRT-PCR (E), and Western blot (F) were employed to examine the indicated genes expression; G–I) transfected HepG2-NTCP cells were treated with 1,000 U/ml IFN-α for 48 h after transfection with wild-type or si-GBP1 plasmids: ELISA kits (G), qRT-PCR (H), and Western blot (I) were employed to examine the indicated genes expression. Data are presented as means ± SD; *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001.

Fig. 5.

GBP1 influenced the expression and distribution of HBsAg. Endogenous localization of HepG2 2.15 cells was detected by immunofluorescence labeling with monoclonal anti-HBsAg (green) and anti-GBPl (red) antibodies. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.
GBP1 influenced the expression and distribution of HBsAg. Endogenous localization of HepG2 2.15 cells was detected by immunofluorescence labeling with monoclonal anti-HBsAg (green) and anti-GBPl (red) antibodies. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.

Fig. 6.

Prediction and verification of binding sites between HBsAg and GBP1. A) The molecular docking technique was used to predict important amino acid sites for the interaction of three sub-proteins of HBs with hGBP1; B) mutants with only the top six amino acid sites with the same docking site in the three groups were introduced into HepG2 2.15 cells. Cellular HBV total RNA, 3.5 kb RNA, and preCRNA expression levels were determined by qRT-PCR; C) the four hGBP1 amino acid mutants (R48A, S73A, Q137A, and D184N) screened were transfected HepG2 2.15 cells. Endogenous localization of HepG2 2.15 cells was detected by immunofluorescence labeling with monoclonal anti-HBsAg (green) and anti-GBP1 (red) antibodies. Nuclei were counterstained with DAPI (blue), scale bar: 50 μm; Data are presented as means ± SD. D) HepG2 2.15 cells were transfected with four hGBP1 amino acid mutants (R48A, S73A, Q137A, and D184N) for 48 h. The expression of HBsAg was analyzed by ELISA kits; E–F) representative Western blot of HBs in the nucleus and cytoplasm of HepG2 2.15 cells transfected with four hGBP1 amino acid mutants (R48A, S73A, Q137A, and D184N) for 48 h. Data are presented as means ± SD.
Prediction and verification of binding sites between HBsAg and GBP1. A) The molecular docking technique was used to predict important amino acid sites for the interaction of three sub-proteins of HBs with hGBP1; B) mutants with only the top six amino acid sites with the same docking site in the three groups were introduced into HepG2 2.15 cells. Cellular HBV total RNA, 3.5 kb RNA, and preCRNA expression levels were determined by qRT-PCR; C) the four hGBP1 amino acid mutants (R48A, S73A, Q137A, and D184N) screened were transfected HepG2 2.15 cells. Endogenous localization of HepG2 2.15 cells was detected by immunofluorescence labeling with monoclonal anti-HBsAg (green) and anti-GBP1 (red) antibodies. Nuclei were counterstained with DAPI (blue), scale bar: 50 μm; Data are presented as means ± SD. D) HepG2 2.15 cells were transfected with four hGBP1 amino acid mutants (R48A, S73A, Q137A, and D184N) for 48 h. The expression of HBsAg was analyzed by ELISA kits; E–F) representative Western blot of HBs in the nucleus and cytoplasm of HepG2 2.15 cells transfected with four hGBP1 amino acid mutants (R48A, S73A, Q137A, and D184N) for 48 h. Data are presented as means ± SD.

Fig. 7.

GBP1 expression in the peripheral blood of patients treated with Peg-IFNα-2b and healthy controls. A–B) Relative expression levels of GBP1 in Peg-IFNα-2b-treated patients and healthy controls. Cellular GBP1 mRNA expression levels were determined by qRT-PCR (A). Serum levels of GBP1 (B) were detected by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C) Correlation between GBP1 levels and Peg-IFNα-2b efficacy using Spearman’s correlation analysis in Peg-IFNα-2b-treated patients. Data are presented as means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
GBP1 expression in the peripheral blood of patients treated with Peg-IFNα-2b and healthy controls. A–B) Relative expression levels of GBP1 in Peg-IFNα-2b-treated patients and healthy controls. Cellular GBP1 mRNA expression levels were determined by qRT-PCR (A). Serum levels of GBP1 (B) were detected by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C) Correlation between GBP1 levels and Peg-IFNα-2b efficacy using Spearman’s correlation analysis in Peg-IFNα-2b-treated patients. Data are presented as means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 8.

GBP1 expression and its diagnostic and prognostic value in non-response and response patients. A–B) Relative expression levels of GBP1 in non-response and response patients. qRT-PCR was performed to detect GBP1 mRNA expression (A). Serum levels of GBP1 (B) were detected by ELISA; C) ROC curve for evaluating the diagnostic and prognostic value of GBP1; D) ROC curve for evaluating the diagnostic and prognostic value of GBP1 in combination with other indicators. Data are presented as means ± SD.
GBP1 expression and its diagnostic and prognostic value in non-response and response patients. A–B) Relative expression levels of GBP1 in non-response and response patients. qRT-PCR was performed to detect GBP1 mRNA expression (A). Serum levels of GBP1 (B) were detected by ELISA; C) ROC curve for evaluating the diagnostic and prognostic value of GBP1; D) ROC curve for evaluating the diagnostic and prognostic value of GBP1 in combination with other indicators. Data are presented as means ± SD.

Clinical characteristics of patients with different effects.

Group Non-response Response
Untreated Treated for 3 months Treated for 6 months Untreated Treated for 3 months Treated for 6 months
Cases 14 20
Age (years) 42.14 ±9.09 39.65 ± 9.69
Sex (M/F) 8/6 9/11
Serum HBsAg level (IU/ml) 2560.04 (1207.33, 6259.34) 1732.10 (801.27, 3413.51) 1942.69 (1257.71, 3423.27) 1133.55 (150.81, 3840.16) 380.82 (23.77, 1509.33) 102.30 (1.38, 199.06)
ALT (U/l) 30.50 (16.25, 44.50) 49.50 (33.75, 86.50) 37.50 (18.25, 6.50) 18.30 (14.00, 27.75) 63.00 (34.25, 80.00) 32.00 (21.25, 50.00)
AST (U/l) 23 (16.50, 33.75) 45.00 (29.50, 76.25) 27.00 (19, 52) 22.00 (18.00, 27.25) 50.00 (33.25, 84.00) 39 (19.75, 48.50)
WBC (109/ml)* 6.01 ± 1.47 3.17 ± 1.06 3.87 ± 1.57 5.55 ± 1.24 3.38 ± 1.28 4.73 ± 3.77
TB (μmol/l) 13.19 ±7.94 11.21 ±4.51 10.40 ±4.45 12.12 ±5.17 11.25 ±4.12 9.12 ±3.39
DB (μmol/l) 4.54 ± 1.77 5.09 ± 1.45 4.14 ± 1.21 4.45 ± 1.43 5.51 ± 1.91 4.51 ± 1.51
Serum GBPI level (ELISA) 1.97 ± 0.31 1.76 ± 0.40 1.73 ± 0.34 2.01 ± 0.31 2.06 ± 0.37 2.10 ± 0.37
PBMC GBPI level (PCR) 1.41 (1.11, 1.84) 1.77 (1.00, 3.63) 2.98 (2.58, 4.58) 1.35 (0.94, 2.72) 6.97 (4.70, 13.27) 5.63 (4.49, 10.89)

Clinical characteristics of patients.

Group Health control CHB (Untreated) CHB (Treated for 3 months) CHB (Treated for 6 months) CHB (Treated for 6 months)
Cases 17 34 34 34 34
Age (years) 26.12 ± 4.30 40.68 ± 1.61 40.68 ± 1.61 40.68 ± 1.61 42 ± 10.12
Sex (M/F) 9/8 17/17 17/17 17/17 22/12
Serum HBsAg level (IU/ml) NA 1785.85 (384.09, 5282.87) 1018.02 (61.53, 2682.86) 197.855 (17.11, 2108.22) NA
ALT (U/l)**** 19.41 ± 5.41 27.17 ± 2.75 61.21 ± 5.89 44.44 ± 5.95 38 (19, 52)
AST (U/l)**** 17.65 ± 5.67 25.05 ± 1.61 65.00 ± 8.67 39.68 ± 4.43 27 (21.75, 45.75)
WBC (109ml)**** 3.55 ± 0.86 5.74 ± 0.23 3.29 ± 0.20 3.89 ± 1.64 5.10 ± 1.44
TB (μmol/l)* 4.96 ± 1.22 12.56 ± 1.09 11.24 ± 0.73 9.65 ± 0.66 10.3 (8.23, 13.03)
DB (μmol/l)**** 2.37 ± 0.88 4.49 ± 0.27 5.34 ± 0.30 4.36 ± 0.24 4.47 ± 1.80
Serum GBPI level (ELISA)**** 1.31 ± 0.23 1.99 ± 0.30 1.94 ± 0.41 1.95 ± 0.40 1.94 ± 0.34
PBMC GBPI level (PCR)*** 2.24 ± 0.53 1.82 ± 0.24 7.89 ± 1.76 5.87 ± 0.64 3.46 (2.47, 5.74)
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